Peripheral microvascular parameters in the nephrotic syndrome

DAVID M. LEWIS, JOHN E. TOOKE, MARTIN BEAMAN, JOHN GAMBLE, and ANGELA C. SHORE

Renal Unit, Royal Devon and Exeter Hospital, and Department of Vascular Medicine, University of Exeter, Exeter; and Department ofPhysiology, Charing Cross & Westminster Medical School, London, England, United Kingdom

Peripheral microvascular parameters in the nephrotic syndrome.Background. Peripheral edema, in combination with severe

proteinuria and low serum albumin levels, is pathognomonic ofthe nephrotic syndrome, yet the exact mechanism of its formationis unknown. Two of the most important of the factors in Starling’sforces controlling fluid filtration across the capillary have hithertonot been studied in nephrotic subjects.

Methods. The hydrostatic capillary pressure at the finger nail-fold in actively nephrotic subjects and age and sex matchedcontrols was studied, using direct puncture of the apex of thecapillary under video microscopy, and a servonulling apparatus togive a direct measurement of capillary pressure. Capillary filtra-tion capacity (CFC) at the calf was measured noninvasively by amodern derivative of the technique of mercury strain gaugeplethysmography. Fifteen nephrotic subjects with a variety ofunderlying pathological lesions, and age matched controls werestudied.

Results. Contrary to the assumption of the “overflow” hypoth-esis of edema formation, there was no evidence of capillaryhypertension. The capillary pressure showed no difference be-tween nephrotic subjects and controls: median (range) of 17.6(12.0 to 24.2) compared with 17.3 (9.0 to 21.6) mm Hg, P 5 NS.CFC was significantly higher in nephrotic subjects than controls[5.23 (3.28 to 8.52) 3 1023 versus 3.55 (2.43 to 5.28) 3 1023

ml/min/100 g/mm Hg, P , 0.01].Conclusions. An increase in CFC provides a potentially novel

mechanism contributing at least in part to the formation ofperipheral edema in the nephrotic syndrome.

The mechanisms of edema formation in the nephroticsyndrome are not resolved at a microvascular level. Theearly underfill theory suggested a physiological process ofrenal salt and water retention, secondary to hypovolemiafrom reduced serum colloid osmotic pressures due tohypoalbuminemia [1]. The stimulus for edema formationwould initially be low serum oncotic pressure and subse-quently salt and water overload [2]. However, an alterna-tive “overflow” hypothesis was developed based on the

findings in several studies that plasma volume is notnecessarily reduced, and indeed is normal or elevated in themajority of subjects [3]. In addition, renal sodium and waterhandling mechanisms have been shown to be impaired inthe nephrotic state in experimental models [4], and humans[5]. The “overflow” hypothesis regards the primary intra-renal deficits in sodium/water excretion to be causing fluidretention, a tendency towards increased plasma volume,and overflow edema formation [6, 7]. At a microvascularlevel, an increase in capillary hydrostatic pressure is hy-pothesized as the factor causing extravascular fluid accu-mulation and edema formation.

Microvascular fluid exchange is governed by the so-calledStarling’s forces. Expressed in equation form for a singlecapillary the flow of fluid across the capillary wall, asemi-permeable membrane, is related to the balance ofhydrostatic and oncotic forces acting across the capillary.

Jv 5 LpS {(Pc 2 Pi) 2 s(pp 2 pi)}

where Jv is the fluid flux or net rate of fluid movement, Lpis the hydraulic conductivity and S the surface area of thecapillary wall; LpS is hydraulic conductance; s is thereflection coefficient for protein; Pc and Pi are the capillaryand interstitial hydrostatic pressures, respectively; and ppand pi are the osmotic pressures of plasma and interstitialfluid, respectively.

The Starling equation applies to a single short section ofcapillary. For a whole organ, including human limbs, thecapillary filtration coefficient or capacity (CFC) representsthe sum of the product of the average hydraulic conductiv-ity of the walls of the exchange vessels within the tissue andtheir total exchange area S(LpS) [8].

A number of the factors in this equation have beenmeasured in the nephrotic state. Colloid osmotic pressureof plasma and interstitial fluid drops in parallel untilprofoundly low levels of serum albumin are reached, animportant anti-edema mechanism [9]. Interstitial hydro-static pressure was elevated in edematous areas as would beexpected [10]. However, the rate limiting factor for micro-vascular fluid exchange, capillary filtration capacity, has notbeen measured, nor has capillary hydrostatic pressure.

The currently favored overflow theory suggests an ele-vated capillary hydrostatic pressure as the driving force

Key words: capillary pressure, capillary filtration capacity, nephrotic,edema, glomerulonephritis, diuretics, Starling forces.

Received for publication January 23, 1998and in revised form May 13, 1998Accepted for publication May 13, 1998

© 1998 by the International Society of Nephrology

Kidney International, Vol. 54 (1998), pp. 1261–1266

1261

resulting in peripheral edema formation. Alternatively,systemic changes in capillary permeability to water [11] andalbumin [12] have been postulated in the nephrotic syn-drome, with increased peripheral transcapillary escaperates or gastrointestinal loss of albumin [13]. The aims ofthe present study were (1) to test the overflow hypothesis,that the capillary hydrostatic pressure of peripheral capil-laries is increased in the nephrotic syndrome; (2) to test thehypothesis that peripheral capillary fluid leakiness is in-creased in the nephrotic syndrome; and (3) to assesswhether capillary pressure and capillary filtration capacityare altered by diuretic therapy.

METHODS

Fifteen subjects (8 females, aged 21 to 79 years) wererecruited. All were clinically and biochemically nephroticwith varying degrees of edema. Details of subjects andcontrols are given in Table 1. Underlying renal pathologieswere as follows: three minimal change glomerulonephritis(MCGN), one later developing focal segmental glomerulo-sclerosis (FSGS), four membranous glomerulonephritis,one myeloma, two amyloid, three proliferative glomerulo-nephritis, one light chain disease, and one undiagnosed(failed renal biopsy). Capillary pressure was measured ineight subjects, the estimation not being possible in theremainder because of: poorly visible capillaries (N 5 1);Raynaud’s phenomenon, which may have a confoundinginfluence on capillary pressure (N 5 2); or inability toschedule the examination before commencing disease mod-ifying treatment (N 5 4). Control subjects were matchedfor age (6 5 years), sex and in the case of premenopausalfemales, menstrual cycle phase. Controls for capillary pres-sure studies were also matched for nailfold skin tempera-ture as capillary pressure is known to be related to thisvariable.

Subjects were asked to refrain from taking caffeine orsmoking for two hours before the tests. Investigations took

place in a resting supine position in a temperature con-trolled environment (22 6 1°C) after acclimatizing supinefor 30 minutes. Brachial arterial blood pressure was deter-mined from five measurements by an automated bloodpressure recorder (Dynamap 845; Critcon Inc., Tampa, FL,USA) and skin temperature measured at the finger nailfoldor calf by thermocouple (Fluke 52; RS Components, Corby,UK). Capillary pressure and filtration coefficient measure-ments were undertaken at separate times but under thesame experimental conditions.

Ten patients were treated with loop diuretics that wereomitted on the day of study. One nephrotic patient hadreceived a short course of nifedipine and this was discon-tinued two days before study. The group of patients inwhom capillary pressure measurements were made did notcontain any with a diagnosis of amyloid, myeloma or lightchain disease.

The study was approved by the local Medical ResearchEthics Committee and participants gave written informedconsent.

Measurement of capillary pressure

The method of capillary pressure measurement has beendescribed in detail elsewhere [14]. Briefly, a scalpel bladewas used to carefully remove the upper dead layer ofopaque stratum corneum from two fingers of the left handwithout inducing bleeding or inflammation. The fingerunder study was held in a plasticine mould to minimisemovement and a Granuflex ring (Squibb, Hounslow, Mid-dlesex, UK) placed around the nailfold to retain a pool of0.9% saline to aid visualization of the capillary and mea-surement of atmospheric pressure. Nailfold capillary pres-sure was measured following direct cannulation of the apexof the capillary loop by glass micropipette held in amicromanipulator. An electronic resistance feedback ser-vonulling system allowed dynamic pressure recording,

Table 1. Characteristics of nephrotic subjects and controls. Results are expressed as median (range)

All nephroticpatients Controls P

Nephrotic patients withcapillary pressure

measurementControls for capillary

pressure group P

Female/male number 8/7 8/7 2/6 2/6Age years 57 (21–79) 56 (21–77) NS 53 (27–69) 57 (27–70) NSBody mass index kg/m2 27 (23–34) 24 (20–35) NS 27 (23–34) 23 (19–32) NSMean arterial pressure

mm Hg103 (77–131) 104 (86–124) NS 102 (98–131) 97 (91–122) NS

24 Hour urinary protein g 7.1 (3.8–22.4) — 7.1 (3.8–22.4) —Urine protein/creatinine ratio — 67 (38–125) —

NR 0–125Serum creatinine 142 (64–604) 79 (70–120) ,0.01 143 (64–280) 86 (77–110) ,0.05

NR 45–120 mmol/literSerum albumin 24 (15–29) 41 (38–44) ,0.01 24 (17–29) 40 (35–44) ,0.01

NR 30–48 g/literTime edematous weeks 8 (1–24) None 8 (2–20) NoneDiuretics 10 None 6 None

Abbreviations are: NR, normal range; NS, not significant.

Lewis et al: Microvascular studies in nephrotic syndrome1262

which was filtered, digitized and stored on computer forlater off-line analysis.

Previous work has shown capillary pressure determina-tions to be highly reproducible both between capillariesacross the nailfold (coefficient of variation 5.4%) in ninecapillaries measured at the same visit in five individuals,and within the same individual over time (coefficient ofvariation 9.6%) for six measurements over six months [15].For capillary pressure studies, previous control populationdata have shown a population SD for capillary pressure of2.5 mm Hg. Based on this 16 subjects in each group wouldbe required to have a 90% power to show a 3 mm Hgdifference in capillary pressure between groups at a signif-icance level of 5%. The capillaries of the finger nailfoldbelong to the same class of continuous capillaries as foundin skin, connective tissues and skeletal muscle. Develop-mentally this area is an outgrowth of the dorsal skin of thehand. During the measurements pitting edema was ob-served in the tissues of the distal phalanx of adjoiningfingers. Nevertheless, it is important not to assume that themeasurements of the capillary pressure at the finger nail-fold are necessarily representative of all edema formingregions.

Measurement of capillary filtration capacity

Mercury-in-silastic strain gauge plethysmography wasused to measure calf capillary filtration capacity (CFC), asdescribed by Gamble, Gartside and Christ [16]. Thismethod measures changes in calf volume in response to aseries of small pressure increments in a thigh occlusioncuff. The technique has a number of theoretical advantagesover single step techniques [8]. A multiple inlet thighocclusion cuff was rapidly inflated by electric pump, withpressure regulated by a multiple resistance air bleed valve.Serial pressure increments of 8 to 12 mm Hg were made atfive minute intervals to increase lower limb venous pres-sure. Calf swelling rate was recorded by a mercury-in-silastic strain gauge mounted in a temperature compensat-ing holder applied to the upper calf. Once ambient venouspressure was exceeded, each increase in cuff pressureproduced a characteristic response. This comprised aninitial rapid calf volume increase, which was due to filling ofcapacitance vessels (the venous compliance) and could bemodeled with a negative exponential curve with a timeconstant of approximately 15 seconds. The concurrentlinear phase to the response reflected the fluid flux acrossthe capillary bed. Traces were recorded on microcomputerfor later analysis. Values for fluid flux were obtained fromthe gradient of the linear phase of each volume responsecurve at a period greater than five time constants from theincrease in cuff pressure. The slope of the regression line offluid flux and corresponding cuff pressure was calculated togive the CFC. The horizontal intercept was the isovolumet-ric venous pressure (Pvi), defined as the pressure at whichthere was neither net fluid filtration nor absorption at the

microvascular wall. Although no net filtration was takingplace, this did not preclude simultaneous movement offluid into and out of the capillary. Calf tissue compliancewas calculated from the gradient of the regression line ofthe venous compliance volumes plotted against cuff pres-sure.

Using this technique to measure capillary filtration coef-ficient in four normal individuals, studied four to six timesover 10 months, the mean intra-individual coefficient ofvariation was 9.1 6 5.6% (mean 6 SD) and for Pvi was10.7 6 4.7%.

Statistical analysis

Data are presented in the text as median (range),assuming a non-parametric distribution. Comparison be-tween groups was made using Mann-Whitney U-test. Cor-relations were examined by the two-tailed Spearman ranktest.

RESULTS

Capillary pressure showed no difference between ne-phrotic subjects and controls: 17.6 (12.0 to 24.2) comparedwith 17.3 (9.0 to 21.6) mm Hg, P 5 NS (Fig. 1). CFC wassignificantly higher in nephrotic subjects than controls: 5.23(3.28 to 8.52) 3 1023 versus 3.55 (2.43 to 5.28) 3 1023

Fig. 1. Capillary pressure in patients with nephrotic syndrome and age-and sex-matched controls. P 5 NS.

Lewis et al: Microvascular studies in nephrotic syndrome 1263

ml/min/100 g/mm Hg, P , 0.01 (Fig. 2). Pvi was significantlylower in nephrotic subjects 14.0 (1.8 to 38.4) versus 24.9(17.7 to 35.6) mm Hg P , 0.01. Calf tissue compliance 5.3(2.8 to 7.1) 3 1022 versus 4.1 (2.5 to 10.2) ml/mm Hg andmean arterial blood pressure 102 (77 to 131) versus 104 (86to 124) mm Hg were the same for both groups (P 5 NS).

CFC results for the nondiuretic treated subgroup aloneshowed this group to have a significantly higher resultscompared to their respective age and sex matched controls:5.17 (4.04 to 6.84) 3 1023 versus 2.92 (2.63 to 4.02) 3 1023

ml/min/100 g/mm Hg, P , 0.05. CFC for the diuretictreated subgroup was 5.52 (3.28 to 8.52) 3 1023 ml/min/100g/mm Hg. Excluding subjects with myeloma, amyloid orlight chain disease, nephrotic subjects had a CFC of 5.18(3.20 to 8.14) 3 1023, and controls of 3.55 (2.63 to 5.28) 31023 (P , 0.01). There was no significant differencebetween capillary pressure in this subgroup and theircontrols, or any significant correlation at a level of signifi-cance of 5% between serum albumin concentration orurinary albumin loss and CFC or capillary pressure. Therewere no evident differences in microvascular parametersbetween the groups of nephrotic subjects with differenthistological diagnoses, neither was there any correlationbetween CFC and renal function as measured by serumcreatinine concentrations.

DISCUSSION

This study has demonstrated an increase in calf capillaryfiltration capacity in patients with active nephrotic syn-drome, as demonstrated by a multi-step strain gauge pleth-ysmography technique, providing a novel explanation foredema formation in this condition. There was no differencein finger nailfold capillary pressure in the subset in whommeasurement was possible, a finding that is at odds with theoverflow theory of edema formation.

Cardiac edema is typically associated with central venouspressure elevation and the traditional model of the patho-genesis suggests that the principal change in Starling’sforces is an elevation of capillary hydrostatic pressure.Indeed, in established peripheral edema states of acutenephritis and cardiac failure [17, 18] finger nailfold capil-lary pressure has been shown to be significantly elevated,albeit with a less accurate method. It is possible thatnephrotic patients with severely diminished renal functionand hypertension might have elevated capillary pressures.In the nephrotic syndrome two models of edema formationhave evolved. The underfill model assumes intravasculardepletion due to fluid flux from plasma caused by depletedplasma oncotic pressure. The relative hypovolemia pro-duced would stimulate a secondary, physiological, renalfluid retention. This hypothesis has been disputed becauseof lack of agreement in studies of plasma volume, assummarized by Dorhout Mees, Geers and Koomans [3].More recently, generalized salt and water overload hasbeen attributed to an intrinsic renal defect causing fluidretention (overflow model), and an increase in capillaryhydrostatic pressure has been proposed to explain theproduction of peripheral edema.

Referring back to the Starling equation, the microvascu-lar basis of edema formation can be considered fromchanges of the various forces. Our data suggest that,contrary to the overflow theory, an elevation of capillarypressure was not involved in edema formation at least atthe time of study, although it could be argued that in thecurrent study patients were examined at a time of re-established steady state equilibrium, with edema accumu-lation at an end. This would be compatible with normalcapillary pressure, but only if other factors were abnormalto maintain the edematous state. Increased fluid flux acrossthe capillary due to increased hydraulic conductance, inkeeping with our findings, could contribute both to forma-tion and maintenance of peripheral edema. Plasma andinterstitial colloid osmotic pressures (pp and pI) fall inparallel as a function of dilution and washout in theinterstitium [9], and hence would not contribute to edema.Interstitial pressure (Pi) is known to have a nonlinearrelationship with interstitial volume [19], and for the degreeof edema seen in the subjects described here the interstitialpressure would be expected to be elevated from the normalmildly negative pressures described in most subcutaneous

Fig. 2. Capillary filtration capacity in patients with nephrotic syndromeand age- and sex-matched controls. P , 0.01.

Lewis et al: Microvascular studies in nephrotic syndrome1264

tissues and species examined [20]. This, and the limitedcompliance of the interstitium found in the early stages ofedema formation, provide an anti-edema mechanism. Thiswould oppose fluid flux due to the increased CFC observedin the present study, once the level of edema has reached asteady state. Only after the orientation of the interstitialproteoglycan structures has been deranged by significantvolume increase does the pressure volume curve adopt aflat alignment and present relatively less impediment tofluid flux from the capillary. Specific impairment of lym-phatic drainage exacerbates the edema rather than beingthe primary cause [21].

The possibility of a circulating factor altering peripheralcapillary permeability in renal disease could be hypothe-sized. Various factors largely peculiar to MCNS or therelated FSGS, such as an altered charge on albumin anderythrocytes [22, 23], have implications for capillary func-tion. Circulating factors affecting glomerular permeabilityhave been proposed for some time [24]. Serum or plasmafrom patients with focal segmental glomerulosclerosis(FSGS) have produced proteinuria in animal models [25],and plasma protein adsorption decreases proteinuria inrecurrent disease after renal transplantation [26]. Vascularpermeability factor (vascular endothelial growth factor) is adisulfide-linked dimeric glycoprotein of about 40 kD thatpromotes fluid and protein leakage from blood vessels [27].It has become a candidate agent in changes of glomerularand possibly systemic permeability in the nephrotic syn-drome, but it appears to be implicated predominately inminimal change nephrotic syndrome (MCNS) and possiblyIgA nephropathy [28, 29]. Few studies discuss these factorswith regard to other renal pathologies, but those that dotend to consider nephrotic syndrome secondary to MCNSand/or FSGS as a separate entity to other nephrotic states[25]. Our study shows no clear trend in the small number ofsubjects with minimal change disease. A unifying hypothe-sis of circulating agent(s) altering vascular permeability inthe nephrotic state remains unproven at present.

With regards to methodology, the changes in CFCobserved in our study imply an alteration of capillaryhydraulic conductivity, or of the functional capillary surfacearea available for exchange. Back perfusion of all availablecapillaries in this technique due to venous distension wouldargue against an increased capillary surface area being anexplanation of the findings [16], although the surface areafor exchange may not be identical to the number ofperfused capillaries: for example, there may be an increasein membrane porosity per unit surface area of capillarywall. Hence, both the effective area of exchange and Lp aresubject to variation. While there is no evidence of increasedtotal capillary surface area in the lower limb of nephroticsubjects, such an increase cannot be ruled out. The pres-ence of edema in the limb being measured would decreasethe ratio of capillary volume/tissue volume that might resultin an underestimation of CFC due to a proportionately

smaller capillary surface area per unit volume than in anonedematous state. The assessment of dry weight inedematous nephrotic subjects is complicated by the under-lying loss of dry body wt [3], probably due largely to loss ofmuscle mass. This would increase the ratio of other tissues,including bone, to the muscle capillary bed and tend toresult in an underestimation of CFC, independent ofchanges in hydraulic conductance. As both factors may leadto underestimates of CFC, they cannot explain our findingsof an increase in CFC in the nephrotic individuals. How-ever, they do raise the possibility that our values mayunderestimate the true values.

Diuretics have been implicated in alteration of albuminpermeability in a rat model [30]. It is unknown whether theuse of loop diuretics in human subjects alters peripheralvascular leakiness. Ten of the subjects in this study hadingested loop diuretics by mouth the day before the tests. Itwas felt unethical to omit them for a longer period beforestudy in patients with active and significant fluid retention.Nevertheless, analyzing the CFC results for the nondiuretictreated subgroup alone shows this group to have a signifi-cantly higher results compared to their respective age andsex matched controls. A number of the nephrotic subjectshad renal impairment, shown by an elevated serum creati-nine concentration. However, there was no correlationbetween serum creatinine and CFC. The possibility ofcomparison with subjects with impaired renal function(rather than normal control subjects) was addressed. Thepossibility of introducing other factors that might effectendothelial function such as hypertension, medications andunderlying renal disorders, as well as uremia per se was feltto be too great to use such a control group. In fact, thedegree of dysfunction present in the nephrotic subjects maynot be as high as suggested by the data in Table 1: only twosubjects had a serum creatinine greater than 200 mmol/liter.

In conclusion, active nephrotic syndrome is associatedwith an increase in systemic CFC, providing a potentiallynovel mechanism for the edema formation characteristic ofthis condition. In contrast, no evidence for capillary hyper-tension was obtained. We are unable to exclude the possi-bility that an elevation in capillary pressure may play a rolein the initiation of edema formation; further studies arerequired to delineate the relative importance of thesemicrovascular parameters at different stages of the diseaseprocess.

ACKNOWLEDGMENTS

This work was partially funded by the Wellcome Trust, referencenumbers 032627/Z/90/Z/1.27/LA and 039501/Z/93/Z/DG. A portion of thispaper was presented at the 1996 meetings of the Renal Association (UK)in Oxford and the EDTA in Amsterdam.

Reprint requests to Dr. David M. Lewis, Renal Unit, Manchester RoyalInfirmary, Oxford Road, Manchester, M13 9WL England, United Kingdom.E-mail: David.Lewis@dial.pipex.com

Lewis et al: Microvascular studies in nephrotic syndrome 1265

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